Genetic variation and population status of sharks: A comparative mtDNA study in the waters of Aceh Jaya and West Aceh

Sharks are one of the keys for ecosystems balance in the ocean. Sharks as top predators have an important role to maintain the stability of the food chain in the ocean. The loss of sharks will have a major impact on the marine ecosystem, so the presence of sharks in the ocean must be protected from extinction. However, there are some activities such as sharks hunting and trading going on because of the high demand of sharks processed products. Bali is one of the locations of shark fishing and trading activities. The problem is the trade-in sharks have gone through the finning process, leaving only the body parts or fins which resulted in the difficulty of morphological identification process. An alternative method that can be used if molecular identification cannot be done is DNA Barcoding. DNA Barcoding means identifying all the animal species with an effective molecular approach applied to identify species. The identification results of DNA Barcoding of shark’s trade in Bali shows that there are four species of sharks have been sold, which is hammerhead sharks (Sphyrna lewini), thresher sharks (Alopias pelagicus), big eye thresher shark (Alopias superliciosus), silky sharks (Carcharinus falciformis). These sharks registered in the IUCN red list, Sphyrna lewini classified as threatened species, while the others (Alopias pelagicus, Alopias superciliosus and Carcharinus falciformis) classified as vulnerable species. In the international trade regulations on CITES, these four species come under the Appendix II. In national regulations, Alopias pelagicus, Alopias superciliosus, Sphyrna lewini have been regulated by the Ministerial regulations, while for Carcharinus falciformis, there are no national regulations yet.

Hammerhead sharks (Sphyrnidae) of southeast Queensland: habitat and movements

Shark is a cartilaginous fish that is widely hunted because it has high economic value. The waters of Aceh are directly adjacent to the Indian Ocean and the Malacca Strait making it a preferred habitat for pelagic fish, especially sharks. Information on shark species landed in the waters west and east of Aceh is very limited due to difficulties in identification and commonly used local names. This study aimed to determine the types of sharks landed in Banda Aceh, Meulaboh, Langsa, Southwest Aceh and Simeulu and to find out specifically what species of sharks are most often landed in Aceh waters. Primary data gathering consisted of four stages, namely sample collection, identification using identification books, molecular identification cytochrome oxidase subunit I (COI) and phylogenetic analysis. The results of the molecular analysis of 46 tissue samples from five locations identified 13 species of sharks, namely Carcharhinhus sorrah, Carcharhinhus amboinensis, Triaenodon obesus, Isurus oxyrinchus, Sphyrna zygaena, Sphyrna lewini, Loxodon macrorhinus, Hemipristis elongaria, Stagostoma fasciatum, Nebrius ferrugineus, Chilloscyllium punctatum, Isurus oxyrinchus, Alopias pelagicus, Alopias supercillosus and 1 species of rays, namely Rhynchobatus australiae. Phylogenetic tree reconstruction using the Neighbor Joining method of 610 basepairs consisting of two large clades separates the species Alopias pelagicus and Isurus oxyrichus with Carcharhinus sorrah, Sphyrna lewini, Loxodon macrorhinus and Rhyncobatus australiae with boostrap values of 87% and 64%. The haplotype diversity shown ranged from 0.667-0.889 while the nucleotide diversity ranged from 0.001-0.097. These values indicates high diversity because of the variance in the number of species found.Keywords:BarcodeCOISharks catchAlopias supercillosusSphyrna lewini

Stored product psocids (Insecta: Psocoptera) is one kind of common storage insect pests in the world and is difficult to be identified morphologically. The molecular identification methods, from Restriction Fragment Length Polymorphism (RFLP), specific primer, to DNA Barcoding, were studied gradually in this paper with international collaboration and based on sequence of 16S rDNA gene of the common species of stored psocids such as Liposcelis. bostrychophila, L. brunnea, L. corrodens, L. decolor, L. entomophila, L. mendax, L. paeta, L. pearmani, L. rufa, and L. tricolor. The samples of different geographical populations in this study were from Peoples Republic of China, Czech Republic, United States of America, Croatia, Portugal and Denmark respectively. The result presented that it was successful to accomplish the sequencing of 16S rDNA gene from single individual of above psocids with one pair of primers (16Sar and 16Sbr) and our methods of DNA extraction, Polymerase Chain Reaction (PCR) reaction mix and condition. Twenty-two sequences were submitted to GenBank and the accession numbers were allocated (Table 1). According to the related sequences, RFLP method was studied firstly and one restrictive endonuclease (DraI) was selected to discriminate the most common four species such as L. bostrychophila, L. entomophila, L. decolor, and L. paeta. The method of specific primer was researched in order to determine L. corrodens which species had not occurred in China. Two pairs of specific primers were designed and selected for the identification of L. corrodens with specific bands from other species of common stored psocids. As an applied example, the samples of Liposcelis captured from imported graze seeds of Denmark by Chinese plant quarantine agency were identified with method of DNA Barcoding. The results showed that Denmark sample shared 98.94% sequence similarity with L. corrodens, and the maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimonious (MP) phylogenetic analysis indicated that Denmark sample and L. corrodens were in the same subgroup in the phylogenetic relationship tree. For further development, we are researching the DNA Barcoding with mtDNA COI (mitochondrial cytochrome oxidase I) gene of stored psocids as the prototype of other stored pests. More collaboration from the world is regarded as the key point for the successful research of molecular identification of stored insect pests.

Several methods of DNA extraction, coupled with ‘DNA barcoding’ species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages – eggs, the first instar larvae, and empty pupal cases (puparia) – using four different extraction methods, namely, one simple ‘homemade’ extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types – eggs, larvae, and pupari.

A constructive step towards selecting a DNA barcode for fungi

The global decline of shark populations, largely driven by overfishing to supply the shark fin trade, poses a significant threat to marine ecosystems. Southeast Asia, and particularly Singapore, is a key hub for the transit and trade of shark fins that contribute to the exploitation of these apex predators. Through the use of DNA barcoding techniques, this study aimed to determine what species of shark are involved in the Singapore shark fin trade. Fins were collected from markets, dried goods shops and traditional Chinese medicine halls throughout Singapore. In total, DNA was extracted from 684 fins collected in January 2024 and PCR amplification targeted a fragment of the mitochondrial COI gene for species identification. Results revealed fins from 24 species across 16 genera, with 19 species listed on CITES Appendices II, and 16 listed as threatened on the IUCN Red List (critically endangered = 2, endangered = 4, vulnerable = 10). The top five most frequently identified species were Carcharhinus falciformis, Galeorhinus galeus, Rhizoprionodon oligolinx, Sphyrna lewini and Rhizoprionodon acutus. Of these, four are listed on CITES Appendix II and four are listed as threatened on the IUCN Red List.

Taxonomy: DNA barcodes: recent successes and future prospects

A fundamental goal in biological research is to gain deeper understanding of the ecological and evolutionary processes that drive speciation. Speciation occurs through a complex combination of phenomena that act between and within individuals, across time and space, driven by biotic and abiotic factors. Characterising these drivers is a challenging task that requires powerful analytic tools and strong study systems. Incipient species that are yet to become fully independent groups offer valuable opportunities to understand speciation by exploring it as it progresses. The Eastern Yellow Robin, a widespread Australian passerine, has a discordant pattern of genetic and phenotypic variation that makes it an ideal candidate to study speciation in action. On one hand, according to nuclear genetic markers and plumage colouration, the species varies along its range in a north-south direction. On the other hand, according to mitochondrial genetic markers, the species has a major, species-level disjunction in an east-west direction, resulting into two highly divergent mitochondrial lineages (mitolineages). The species presents a notable example of mitochondrial-nuclear (mitonuclear) discordance: a pattern in which mitochondrial gene and nuclear gene variation have discordant patterns of geographic differentiation. By disentangling this complex pattern of variation and testing hypotheses about causation, I revealed significant events during the Eastern Yellow Robins evolutionary history and proposed a mechanism by which the mitolineages are currently undergoing speciation. The Eastern Yellow Robin started to differentiate more than 1.3 million years ago into two geographically northern and southern isolated populations. During this period, much of the nuclear DNA and plumage colouration differentiation accumulated. Subsequently, these two populations came into secondary contact, generating the current pattern of north-south nuclear genetic and colour variation. After secondary contact, two independent events of adaptive mitochondrial introgression generated the current pattern of east-west mitochondrial genetic variation. According to this scenario, northern mitochondria introgressed southwards along the west and southern mitochondria introgressed northwards along the east. Mitochondrial introgression introduced adaptive genetic variation that assisted west and east mitolineages to thrive in their local environments. However, mitolineages are currently undergoing nuclear gene flow (dispersal and mating), therefore, a more complicated mechanism must be in place that prevents the two mitolineages from becoming unified. Mitochondrial and nuclear genes co-evolve to maintain essential cellular functions, such as gene expression, protein-complex assembly, metabolic, and physiological activities. By scanning thousands of genetic markers along the nuclear genome I found evidence that a subset of nuclear genetic variation accompanies the mitochondrial split. This is consistent with natural selection acting to avoid mitonuclear combinations being disrupted in intermediate forms between west and east mitolineages (i.e. hybrids). The great majority of the highly differentiated nuclear genes between mitolineages are clustered in one large genomic region. This region contains closely-linked, putatively co-adapted genes (i.e. it functions as a supergene) that are related to essential metabolic and physiological functions. This thesis contributes to a growing appreciation that interacting mitochondrial and nuclear genes involved in metabolism can influence the pattern and process of species formation.

DNA barcoding of commercially important groupers (Epinephelidae) in Aceh, Indonesia

The fisheries sector is one of the exellence sectors in the West of Aceh. Generally, mostly fishermen have the trammel net that used in fishing operation. The trammel net is a fishing gear that used in the sea bottom for catching a lot of shrimps. The aiming of this research is to analyse catches composition of the trammel net. The research method that used was an experimental fishing. The results showed that catches composition of trammel net are 14 species including Metapenaeus affinis, Fenneropenaeus merguiensis, Aristaeomorpha foliacea, Harpodon nehereus, Sphyrna lewini, Rastrelliger sp, Selar crumenophthalmus, Caesio chrysozon, Cynoglossus lingua, Thryssa setirostris, Terapon jarbua, Cyclocheilichthys armatus, Leiognathus equulus and Rachycentron canadum. Based on the catches composition are Leiognathus equulus are dominant with 19.37%, Metapenaeus affinis with 15.50%, and Aristaeomorpha foliacea 11.62%. Moreover, the rare species including Hammerheads (Sphyrna lewini) and mackerel (Rastrelliger sp) as much as 0.77%. Sampling result also obtained amount of individuals as much as 129 individuals including 38 species target (shrimps) with 29.45% and non target species as much as 91 with 70.54%.Keywords : Trammel Net, Suak Seumaseh, West Aceh, Catch Composition

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller's classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.

Early detection of economically important insects is critical to preventing their establishment as serious pests. To accomplish this, tools for rapid and accurate species identification are needed. DNA barcoding, using short DNA sequences as species "genetic identification tags," has already shown large potential as a tool for rapid and accurate detection of economically important insects. DNA extraction is the critical first step in generating DNA barcodes and can be a rate-limiting step in very large barcoding studies. Consequently, a DNA extraction method that is rapid, easy to use, cost-effective, robust enough to cope with range of qualities and quantities of tissue, and can be adapted to robotic systems will provide the best method for high-throughput production of DNA barcodes. We tested the performance of a new commercial kit (prepGEM), which uses a novel, streamlined approach to DNA extraction, and we compared it with two other commercial kits (ChargeSwitch and Aquapure), which differ in their method of DNA extraction. We compared performance of these kits by measuring percentage of polymerase chain reaction (PCR) success and mean PCR product yield across a variety of arthropod taxa, whichincluded freshly collected, ethanol-preserved, and dried specimens of different ages. ChargeSwitch and prepGEM performed equally well, but they outperformed Aquapure. prepGEM was much faster, easier to use, and cheaper than ChargeSwitch, but ChargeSwitch performed slightly better for older (> 5-yr-old) dried insect specimens. Overall, prepGEM may provide a highly streamlined method of DNA extraction for fresh, ethanol-preserved, and young, dried specimens, especially when adapted for high-throughput, robotic systems.

The Scalloped Hammerhead, Sphyrna lewini, is a large coastal pelagic shark species that inhabits tropical and subtropical waters around the world. It is listed as Critically Endangered by the International Union for Conservation of Nature (IUCN, Red List). In the present study, we used nine nuclear microsatellite DNA markers and sequences of the complete mitochondrial DNA genome to estimate the diversity and genetic structure of S. lewini in the Gulf of Mexico and to assess whether the genetic evidence supports philopatry within this geographic area. We sampled a total of 73 juvenile individuals from seven locations in the Northern (GMN) and Southern (GMS) Gulf of Mexico. Our results indicate low genetic diversity in the Gulf of Mexico population compared to previously studied populations, which could be related to the origin and colonization of the species. We detected genetic homogeneity in both types of markers, which suggests that philopatric behavior is unlikely in the studied area. Interestingly, the location La Pesca was genetically distinct from the rest of sampled locations, which may warrant special attention for conservation efforts.

The scalloped hammerhead shark, Sphyrna lewini (Griffith and Smith, 1834), is an important species along the Indian coast both in the Arabian Sea and Bay of Bengal. As per the IUCN Red List Assessment, the scalloped hammerhead shark is categorized as “globally endangered.” We investigated the intra-specific genetic diversity and population genetic structure of S. lewini along the Indian coast (Arabian Sea and Bay of Bengal) by analyzing mitochondrial cytochrome C oxidase 1 and control region sequences, respectively. Furthermore, comparisons of the sequence data generated from the present study with the sequences of S. lewini retrieved from GenBank revealed the presence of three (3) haplogroups corresponding to Indian/Indo-Pacific, Atlantic, and Pacific regions. A lack of genetic differentiation within the Indian coast and significant differentiation between ocean basins (FST; 0.80; p < 0.001) indicated female philopatry as mitochondrial DNA is maternally inherited. Hammerhead sharks are inherently vulnerable to fishing practices directly and indirectly (as bycatch) due to their evolutionary distinct features like cephalofoil. Specialized species-specific management measures are urgently needed to protect these precious resources of the oceans.